Journal of Clinical Virology 21 (2001) 197– 212

www.elsevier.com/locate/jcv

Resistance of human immunodeficiency virus type 1 to

reverse transcriptase and protease inhibitors: genotypic and
phenotypic testing
J. Gerardo Garcı́a-Lerma, Walid Heneine *
HIV and Retro6irology Branch, Di6ision of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases,
Centers for Disease Control and Pre6ention, Atlanta, GA 30333, USA

Abstract

Treatment of HIV-1-infected persons with antiretroviral drugs including reverse transcriptase (RT) and protease
inhibitors has significantly reduced the rate of HIV and AIDS-related morbidity and mortality. However, these
treatments can select for drug-resistant viruses which are associated with poor virologic responses to the antiretroviral
therapies and loss of clinical benefit. Drug resistance is conferred by single or several amino acid changes in the pol
gene. These mutations can be classified as primary when they directly confer reduced drug susceptibility, or secondary
when their influence is primarily on replication capabilities of resistant viruses. Both genotypic and phenotypic
methods are used for drug resistance testing. Genotypic assays detect resistance-related mutations by sequence
analysis or point mutations assays. Phenotypic testing measures drug susceptibility of patient-derived viruses in
culture assays. Viruses can be conventionally isolated from peripheral blood lymphocytes, or generated more rapidly
through recombination of plasma-derived RT/protease sequences and modified HIV-1 vectors. Phenotypic testing
provides direct evidence of resistance, is easy to interpret, but is laborious and expensive. In contrast, genotypic
testing provides indirect evidence of resistance, is relatively faster and cheaper, but some complex mutation patterns
may be difficult to interpret. Non-culture based phenotypic assays that measure susceptibility of RT activity in plasma
to RT inhibitors have been described recently, and provide new tools for rapid phenotypic testing. Resistance testing
is currently recommended to help guide the choice of new regimens after treatment failure and for guiding therapy
in pregnant women. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: HIV-1 drug resistance; Phenotype; Genotype

1. Introduction munodeficiency virus type 1 (HIV-1) (Mitsuya et

Since the discovery of zidovudine (ZDV) as an
effective antiretroviral agent against human im-
* Corresponding author. Tel.: + 1-404-6390218; fax: + 1-
404-6391174.
al., 1985), drug therapy has been widely used in
the treatment of AIDS. Combination of antiretro-
viral drugs targeting the reverse transcriptase
(RT) and/or protease of HIV-1 can suppress
plasma levels of the virus and delay disease pro-
gression in HIV-1-infected persons, thus reducing

1386-6532/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 6 - 6 5 3 2 ( 0 0 ) 0 0 1 6 3 - 3
198 J.G. García-Lerma, W. Heneine / Journal of Clinical Virology 21 (2001) 197–212

the rate of HIV and AIDS-related morbidity and HIV-1 RT include nucleoside and non-nucleoside
mortality (Gulick et al., 1997; Hammer et al., RT inhibitors. The nucleoside RT inhibitors (NR-
1997). Despite their ability to suppress virus repli- TIs) class of compounds share a similar mecha-
cation for extended periods of time, current an- nism of action and require intracellular activation
tiretroviral therapies have thus far been unable to by phosphorylation to the 5%-triphosphate form
eradicate HIV-1 from infected persons (Pierson et (Arts and Wainberg, 1996). The triphosphate
al., 2000). form of NRTIs inhibits HIV-1 RT through com-
The clinical benefit of antiretroviral therapy is petitive inhibition with the triphosphate form of
compromised by the emergence of drug-resistant the native deoxynucleoside, and chain termination
variants of HIV-1. Emergence of drug-resistant of elongating DNA (Arts and Wainberg, 1996).
HIV-1 is due to the ability of the virus to escape The six NRTIs approved for treatment are ZDV,
under drug-selective pressures. Virus escape is a didanosine (ddI), zalcitabine (ddC), stavudine
consequence of the population structure of HIV-1 (d4T), lamivudine (3TC) and abacavir (1592U89)
which consists of a complex distribution of related (Mitsuya et al., 1985; Mitsuya and Broder, 1986;
but not identical HIV-1 genomes known as qua- Lin et al., 1987; Yarchoan et al., 1989; Coates et
sispecies (Domingo et al., 1995, 1997, 1998). The al., 1992; Daluge et al., 1997).
quasispecies distribution results from the absence The non-nucleoside RT inhibitors (NNRTIs)
of proofreading-repair activities during RNA-de- are structurally and chemically dissimilar com-
pendent DNA synthesis by the viral RT. Calcula- pounds that are potent inhibitors of HIV-1 RT.
tions of the mutation rate for HIV-1 predict that In contrast to NRTIs, the NNRTIs do not require
each newly copied genome differs from the metabolic activation to manifest their antiviral
parental virus on average by a single nucleotide
(Mansky and Temin, 1995). If an estimated 1010 Table 1
virions are produced daily in an HIV-1-infected Antiretroviral drugs currently approved for treatment of HIV-
person (Perelson et al., 1996) and each genome 1-infected persons
contains an average of 1 mutation, every possible Generic name Trade name FDA approval
single mutation associated with drug resistance
may be generated daily. Drug resistance muta- Nucleoside RT inhibitors
tions have been identified in the RT gene from Zidovudine (ZDV) Retrovir® 1986
patients who have not received antiretroviral Didanosine (ddl) Videx® 1991
Zalcitabine (ddC) Hivid® 1992
drugs (Nájera et al., 1994, 1995; de Jong et al., Stavudine (d4T) Zerit® 1994
1996). Estimates based on the rate of emergence Lamivudine (3TC) Epivir® 1995
of nevirapine resistance in previously untreated Abacavir (1592U89) Ziagen® 1998
persons indicate that approximately 1 in 1000 Non-nucleoside RT inhibitors
genomes carry the Y181C mutation associated Nevirapine Viramune® 1996
with nevirapine resistance before treatment with (BI-RG-587)
nevirapine (Havlir et al., 1996). Therefore, pre-ex- Delavirdine Rescriptor® 1997
(U-90152)
isting viruses with resistance mutations may be-
Efavirenz Sustiva™, 1998
come rapidly selected by antiretroviral drugs. (DMP 266) Stocrin™
Protease inhibitors
Indinavir (MK 639) Crixivan® 1996
2. Inhibitors of HIV-1 reverse transcriptase and Ritonavir (ABT 538) Norvir® 1996
protease Saquinavir Invirase®, 1995, 1997
(RO 31-8959) Fortovase®
To date, a total of 14 antiretroviral drugs have Nelfinavir Viracept® 1997
(AG 1343)
been approved for treatment of HIV-1-infected Amprenavir Agenerase® 1999
persons (Table 1). These drugs target either the (141W94)
RT or the protease of the virus. Inhibitors of
 J.G. García-Lerma, W. Heneine / Journal of Clinical Virology 21 (2001) 197–212 199

effects (Spence et al., 1995; Joly and Yeni, 1999). Table 2

Mutations associated with resistance to HIV-1 RT inhibitors
Inhibition of HIV-1 RT by NNRTIs occurs by
non competitive binding to conserved residues of RT inhibitor Primary mutations Secondary
the p66 subunit of the RT, which alters the con- mutations
formational structure and modify the active site of
the enzyme (Spence et al., 1995; Maga et al., 1997; Nucleoside RT inhibitors
Zidovudine K70R, T215Y/F M41L, D67N,
Joly and Yeni 1999). These compounds are spe-
L210W, K219Q
cific for HIV-1 group M isolates and do not have Didanosine L74V K65R, M184V/I
a significant antiviral effect on HIV-1 group O, Zalcitabine K65R, T69D,
HIV-2, or SIV isolates (Descamps et al., 1995, L74V, M184V/I
1997; Witvrouw et al., 1999). The three NNRTIs Lamivudine E44D, V 118I,
M184V
approved are nevirapine, delavirdine, and efa-
Stavudine V75T
virenz (Wu et al., 1991; Dueweke et al., 1993b; Abacavir K65R, L74V, M41L, D67N,
Young et al., 1995). M184V K70R, Y115F,
The HIV-1 protease inhibitors class of com- L210W, K219Q
pounds are peptidomimetic that bind to the active Multinucleoside Q151M A62V, V75I, F77L,
resistance (a) F116Y
site of the viral protease enzyme. Unlike RT
Multinucleoside T69S-SA/G/S M41L, A62V,
inhibitors, protease inhibitors block the produc- resistance (b) D67N, K70R,
tion of infectious virus from infected cells by L210W, T215Y,
inhibiting the cleavage of precursor polyproteins K219Q
necessary to produce infectious virus particles. Non-nucleoside RT inhibitors
Therefore, virus particles produced by infected Nevirapine K103N, V106A, L100I
cells treated with protease inhibitors contain un- V108I, Y181C,
processed precursors and are non-infectious (Kohl Y188C, G190A
Delavirdine K103N, Y181C P236L
et al., 1988; Peng et al., 1989). The five currently
Efavirenz K103N, Y188L, L100I, V108I,
approved protease inhibitors are saquinavir, indi- G190A P225H
navir, ritonavir, nelfinavir, and amprenavir
(Roberts et al., 1990; Ho et al., 1994; Vacca et al.,
1994; Parteledis et al., 1995; Kaldor et al., 1997). A62V, V75I, F77L, and F116Y mutations in-
crease replication capabilities and level of resis-
tance in viruses carrying the Q151M mutation
3. Mutations associated with drug resistance (Maeda et al., 1998; Kosalaraksa et al., 1999), and
the L63P mutation compensates for the impair-
Mutations associated with resistance can be ment of fitness caused by the saquinavir-selected
classified as primary or secondary. Primary muta- L90M mutation (Martı́nez-Picado et al., 1999). A
tions usually appear early, are relatively specific list of primary and secondary mutations associ-
for each drug, and decrease drug susceptibility. ated with resistance to RT and protease inhibitors
Secondary mutations, also called compensatory is shown in Table 2 and Table 3, respectively.
mutations, usually accumulate in viral genomes
that already have some primary mutations. These
secondary mutations confer little or no reduction 3.1. Resistance to NRTIs
in drug susceptibility by themselves, restore repli-
cation capabilities of virus carrying primary muta- Resistance to NRTIs may result from single
tions, and may also increase the level of resistance mutations or accumulation of several mutations
in such viruses. For instance, the L210W muta- in the pol gene. For instance, the M184V muta-
tion increases the level of ZDV resistance of tion that is associated with resistance to 3TC can
viruses that harbor other ZDV resistant mutations alone reduce the susceptibility for this drug \
(Harrigan et al., 1996; Hooker et al., 1996), the 100-fold (Boucher et al., 1993; Schinazi et al.,
200 J.G. García-Lerma, W. Heneine / Journal of Clinical Virology 21 (2001) 197–212

1993; Tisdale et al., 1993). Other mutations such Resistance mutations to a particular NRTI can
as E44D or V118I can also confer moderate (4– affect the susceptibility of the virus to other
50-fold) levels of 3TC resistance in a background NRTI. For instance, the M184V or L74V muta-
of ZDV resistance mutations (Hertogs et al., tions selected during 3TC and ddI treatment,
2000). High level resistance to ZDV requires the respectively, can transiently suppress phenotypic
accumulation of several mutations (Larder and resistance to ZDV in viruses that have ZDV resis-
Kemp, 1989). The level of ZDV resistance con- tance mutations (St Clair et al., 1991; Larder et
ferred by the K70R and T215Y/F mutations al., 1995). The molecular mechanisms of these
alone varies from 8– 16-fold. In contrast, combi- interactions are not well understood.
nation of these and other secondary mutations A small proportion ( 1%) of patients treated
such as K219Q or L210W decrease ZDV suscepti- with NRTIs develop resistance through a T69S
bility \100-fold (Kellam et al., 1994; Lacey and mutation and a family of insertion mutations
Larder, 1994a). Low level resistance to ddI and (usually two serines) between positions 69 and 70
ddC is associated with the L74V, K65R and of the RT. The presence of this insertion along
T69D mutations (St Clair et al., 1991; Kozal et with the T215Y mutation results in multiple
al., 1994; Zhang et al., 1994). The L74V, K65R, dideoxynucleoside resistance (MDNR) (Winters et
and M184V mutations also confer resistance to al., 1998; de Jong et al., 1999; Larder et al., 1999).
abacavir (Harrigan et al., 2000). The basis of d4T These findings illustrate the ability of HIV-1 to
resistance is not fully clear. Resistance to d4T has escape drug pressure even through amino acid
been associated with insertion mutations at codon insertions in a region of the enzyme that is critical
69, and the Q151M or V75T mutations (Lacey for the polymerase function (Huang et al., 1998).
and Larder, 1994b; Lin et al., 1999). However, Other mutations associated with MDNR are
d4T resistance occurs at low frequency and these A62V, V75I, F77L, F116Y, and Q151M (Shi-
mutations are rarely seen in patients who fail d4T rasaka et al., 1993, 1995; Ueno et al., 1995;
treatment (Lin et al., 1999). Iversen et al., 1996; Schmit et al., 1996; Garcı́a-
Lerma et al., 1999; Van Laethem et al., 2000).
Table 3
These mutations have been observed in 3–16% of
Mutations associated with resistance to HIV-1 protease in-
hibitors patients treated with ZDV and ddC/ddI, and con-
fer resistance to all currently approved nucleoside
Protease Primary Secondary mutations analogs including ZDV, ddI, ddC, d4T, 3TC, and
inhibitor mutations abacavir (Shafer et al., 1994, 1995; Schmit et al.,
Indinavir M46I, L10I/R/V, K20M/R, 1998; Kavlick et al., 1998). The emergence of
V82A/F/T/S L24I, V32I, M36I, these MDNR mutations compromises the clinical
I54V, A71V/T, efficacy of this entire class of compounds, and
G73S/A, V77I, I84V, therefore, severely limits treatment options.
L90M
Ritonavir V82A/F/T/S K20M/R, V32I, L33F,
Development of ZDV resistance in patients
M36I, M46I/L, treated with ZDV+ ddI/ddC is known to occur
I54V/L, A71V/T, V77I, through acquisition of either the T215Y or
I84V, L90M Q151M mutation but very rarely through both
Saquinavir G48V, L90M L10I/R/V, I54V,
mutation (Kavlick et al., 1998; Schmit et al.,
A71V/T, G73S, V77I,
V82A, I84V 1998). The viral determinants that may influence
Nelfinavir D30N, L90M Ll0F/I, M36I, M46I/L, selection of either Q151M or T215Y remain un-
A71V, V77I, defined. However, we have recently found indirect
V82A/F/T/S, I84V, evidence for a role of the viral background and an
N88D
Amprenavir I50V, I84V L10F/I/R/V, V32I,
intermediate mutation (Q151L) in the develop-
M46I, I47V, I54V ment of MDNR mediated by Q151M (Garcı́a-
Lerma et al., 2000a).
 J.G. García-Lerma, W. Heneine / Journal of Clinical Virology 21 (2001) 197–212
3.2. Resistance to NNRTIs
The binding site of NNRTIs is a hydrophobic
pocket close to the polymerase catalytic site in the
p66 subunit of RT. Since these compounds share
a common mechanism of action and bind to a
common site of the enzyme, any slight variation
due to single point mutations may have a signifi-
cant impact on the sensitivity to this class of
compounds. These point mutations are well char-
acterized and result in the loss of stabilizing inter-
actions and the emergence of steric and
thermodynamic barriers for NNRTI binding
(Maga et al., 1997).
NNRTIs select for mutations in two different
regions of the RT (codons 98– 108 and codons
179–190). Resistance to nevirapine is associated
with the A98G, L100I, K103N, V106A, V108I,
Y181C, Y188C, and G190A mutations (Richman
et al., 1994; Havlir et al., 1995; D’Aquila et al.,
1996; Montaner et al., 1998). The Y181C muta-
tion is the most frequently seen mutation in pa-
tients treated with nevirapine monotherapy. This
mutation alone results in \ 100-fold decreased
susceptibility to nevirapine (Richman et al., 1991;
Havlir et al., 1996). Selection of additional muta-
tions associated with nevirapine resistance such as
K103N, Y188C, and G190A is more frequently
observed in patients receiving combination ther-
apy with other drugs such as ZDV (Richman et
al., 1994).
A recent clinical trial done in Uganda has
shown that a single-dose of nevirapine monother-
apy can reduce vertical HIV-1 transmission in
50% during the first 14 – 16 weeks of life compared
with the standard ZDV monotherapy, providing a
simple and inexpensive regimen that can be used
in less-developed countries (Guay et al., 1999).
Preliminary evidence indicate that about one third
of the women developed genotypic resistance to
nevirapine 6 weeks after treatment. However, the
mutation identified in all the women was K103N
and not the previously observed Y181C mutation
(Becker-Pergola et al., 2000). Selection of K103N
may probably be related to the non-subtype B
viral background.
The predominant mutation associated with
delavirdine resistance is the K103N mutation.
201
Other mutations such as Y181C or P236L have
been observed at low frequency alone or in com-
bination with K103N (Dueweke et al., 1993a;
Demeter et al., 1997). The K103N mutation is
also the mutation most frequently seen in patients
who fail treatment with efavirenz. K103N alone
confers moderate resistance to efavirenz (20-fold
reduced susceptibility), and cross resistance to
nevirapine and delavirdine. High level resistance
to efavirenz requires combination of mutations
such as K103N/V108I, K103N/P225H, or
K103N/L100I (Bacheler et al., 1999; Jeffrey et al.,
1999).
3.3. Resistance to protease inhibitors
To date, more than 20 amino acid substitutions
have been observed in the HIV-1 protease follow-
ing treatment with protease inhibitors (Table 2).
Primary mutations associated with resistance to
protease inhibitors are relatively specific for each
drug (e.g. L90M and G48V for saquinavir, or
I50V and I84V for amprenavir). Cross resistance
among protease inhibitors is associated with the
presence of additional secondary mutations (Con-
dra et al., 1995), which may appear later during
treatment or can pre-exist as natural polymor-
phisms (Lech et al., 1996). However, the existence
of two or more primary mutations is likely to
confer broad cross-resistance to most currently
available protease inhibitors (Condra et al., 1995;
Lorenzi et al., 1999).
4. Resistance tests
Resistance of HIV-1 to RT or protease in-
hibitors can be determined genotypically by exam-
ining resistance-related mutations in the viral
genome, and/or phenotypically by measuring the
inhibition of virus replication or enzymatic activ-
ity of RT/protease by specific drugs. To date,
several phenotypic and genotypic assays have
been developed and are being used to monitor for
drug resistance. The relative advantages of geno-
typic and phenotypic assays are shown in Table 4.
202 J.G. García-Lerma, W. Heneine / Journal of Clinical Virology 21 (2001) 197–212
Table 4
Advantages and disadvantages of phenotypic and genotypic assays
Assay Advantages
Phenotypic assays
Replication- Direct measure of drug susceptibility
based assays Can assess complex resistance patterns
Results are more familiar to clinicians
Enzymatic assays Rapid (1–2 days), simple, and cheap
Can detect low proportion of resistant
viruses
Genotypic assays Cheaper and more rapid than replication-
based assays
Less technically demanding than replication-
based assays
4.1. Genotypic assays
All genotypic assays currently used are based
on PCR amplification of viral pol sequences pri-
marily from plasma by RT-PCR. Amplified prod-
ucts can be analyzed by hybridization or
sequencing techniques. Hybridization techniques
are illustrated by the commercially available Line
Probe Assay (LIPA HIV-1 RT, Innogenetics) that
detects key resistance mutations. The LIPA assay
is available as a kit, and is based on reverse
hybridization of biotin-labeled amplified HIV-1
sequences from plasma with short, immobilized
oligonucleotides. The hybrid is detected with a
streptavidine-alkaline phosphatase-based colori-
metric procedure (Stuyver et al., 1997, 1999). The
assay detects both wild type and resistant vari-
ants, and has a high sensitivity in detecting low
proportion (10%) of resistant viruses (Stuyver
et al., 1997). Fig. 1b shows the ability of the assay
to detect 10% of viruses carrying the M184V
mutation in a background of wild type viruses.
However, this method is available for genotyping
only selected RT mutations, and may occasionally
give false negative results due to the presence of
polymorphisms/mutations near the site of the spe-
cific hybridization (Kock et al., 1999; Puchham-
mer-Stockl et al., 1999). In contrast, sequence
Disadvantages
Expensive, laborious
Long time to results (2–4 weeks)
Clinically relevant cutoff values not clearly defined
Rapid protease assays not developed
Currently unable to detect AZT resistance
Useful for subtype B and non-subtype B HIV-1
Indirect measure of susceptibility
Requires expert clinical interprepation
Resistance patterns not fully defined
analysis provides information on all positions as-
sociated with drug resistance, and several compa-
nies are now developing kits including Visible
Genetics and ABI/Perkin Elmer.
Expert clinical interpretation of genotypic re-
sults is important and may often be complicated
by the presence of complex mutations patterns.
Important issues for analysis of genotypic results
also include laboratory quality assurance, and use
of appropriate controls. A recent study that used
well-defined coded samples has shown large inter
laboratory differences in the quality of the results
obtained by automated DNA sequencing in 23
laboratories worldwide (Schuurman et al., 1999).
These findings highlight the need for proper qual-
ity control and standardization among currently
used sequencing methods.
4.2. Phenotypic assays
Phenotypic testing measures the ability of an
HIV-1 isolate to replicate in the presence of a
drug or, alternatively, the enzymatic susceptibility
of RT/protease to inhibition by a drug. Suscepti-
bility of HIV-1 is determined by measuring the
drug concentrations that inhibit in 50 or 90%
virus replication (IC50 and IC90, respectively) or
enzymatic activity. Comparison of the IC50 and
 J.G. García-Lerma, W. Heneine / Journal of Clinical Virology 21 (2001) 197–212 203

IC90 values of a particular virus with those found
in a reference wild type HIV-1, provides a direct
measurement of the level of resistance (i.e. fold
increase in IC50) to the specific drug.
4.3. Replication-based assays
Replication-based assays require the use of
HIV-1 clinical isolates. Conventionally, HIV-1
isolates are obtained from cultures of peripheral
blood lymphocytes or plasma. Drug susceptibility
testing of these viral isolates can then be measured
by several methods including the HeLa CD4+
plaque reduction assay and the peripheral blood
mononuclear cell (PBMC)-based culture system.
The HeLa CD4 + plaque reduction assay was
the first standardized phenotypic test (Larder et
al., 1990). However, this assay is only suitable for
syncytium-inducing (SI) strains, and most patients
isolates contain nonsyncytium-inducing (NSI)
strains or a mixture of SI and NSI strains. The
PBMC-based culture system, in contrast, could be
used in more than 80% of clinical isolates. A
standardized protocol for drug susceptibility test-
ing in PBMCs, called the ACTG-DoD (AIDS
Clinical Trials Group, Department of Defense) is
widely used for drug susceptibility testing (Japour
et al., 1993). However, this assay requires a final
evaluation with an expensive p24 sandwich
ELISA, is time consuming and labor intensive,
and is also fraught with biologic variabilities,
including those related to virus isolation and
tropism.
To circumvent the problem of virus isolation
and cellular tropism, several recombinant virus
assays (RVA) have been developed. A schematic
representation of the assay is shown in Fig. 2.
This assay uses patient-derived RT sequences that
are amplified by PCR and an RT-deleted proviral
laboratory clone to generate recombinant viruses
in a human T cell line by homologous recombina-
tion (Kellam and Larder, 1994). The first RVA
was designed to measure susceptibility to RT
inhibitors only, and used PBMC as a source of
viral sequences (Kellam and Larder, 1994). These
recombinant viruses retain the drug susceptibility
of the RT present in the clinical specimen and can
be cultured in vitro without the selective effect of
virus tropism. The assay was later modified to use
plasma viral RNA, and to allow drug susceptibil-
ity testing to protease inhibitors (Hertogs et al.,
1998).
To date, two RVA are commercially available
to measure susceptibility to RT and protease in-
hibitors: Virco Antivirogram™ and ViroLogic
PhenoSense™. Both assays amplify HIV-1 RT

Fig. 1. Detection threshold of 3TC resistance mediated by the M184V mutation by a phenotypic (Amp-RT) or a genotypic (LIPA)
assay (adapted from Garcı́a-Lerma et al., 1999). (A) Levels of RT inhibition by 3TC-TP measured by Amp-RT in mixtures of wild
type and 3TC-resistant viruses; (B) genotypic detection of the M184V mutation by the HIV-1 LIPA in the same virus mixtures.
204 J.G. García-Lerma, W. Heneine / Journal of Clinical Virology 21 (2001) 197–212
Fig. 2. Schematic representation of the first recombinant virus
assay developed by Kellam and Larder (1994). The assay
allowed the generation of viable viruses by homologous re-
combination of a PCR-derived pool of RT coding sequences
from a patient and an RTdeleted, noninfectious proviral clone.
IC50 and IC90 values for drug were determined using a T cell
line.
and protease but differ in technical aspects related
to recombinant virus construction and detection
of virus replication. Drug susceptibility results are
reported as IC50 values for each drug and fold
differences of these values relative to a reference
wild type virus. Fold differences \4 and \2.5
are considered evidence of decreased susceptibility
for the Antivirogram and PhenoSense assays,
respectively.
The ViroLogic PhenoSense™ assay (ViroLogic,
South San Francisco, CA) measure susceptibility
by using resistance test vectors (RTVs) that con-
tain a luciferase indicator gene and patient-
derived RT and protease sequences (Petropoulos
et al., 2000). Basically, cells transfected with
RTVs produce viral particles that are used to
infect target cells. Since RTVs are replication
defective, luciferase activity is measured after a
single round of viral replication (Petropoulos et
al., 2000). Luciferase activity measured in the
presence of RT and protease inhibitors is used to
measure the level of drug resistance.
The Virco Antivirogram™ assay (Antiviro-
gram; Virco, Mechelen, Belgium) is based on in-
tracellular homologous recombination of
patient-derived gag/RT/protease sequences and a
gag/RT/protease-deleted proviral clone. Replica-
tion of the recombinant viruses is analyzed in the
presence of various concentrations of antiretrovi-
ral drugs using a reporter gene-based cellular
assay (Pauwels et al., 1998).
A comparative analysis of Virco Antiviro-
gram™ and ViroLogic Phenosense™ has been
recently done using coded plasma samples from
drug-naı̈ve or drug-experience HIV-1-infected per-
sons (Qari et al., 2000). The results showed a 92%
concordance between results by both assays, with
the majority of the discordant results observed
among samples that had IC50 values close to assay
cut-off values. These findings indicate that despite
the use of different testing strategies in both as-
says, there is a good correlation between test
results.
While phenotypic data are easy to interpret in
both assays, there are at present no well defined,
clinically validated cut-off values that correlate
virologic response with IC50 values. It may be
reasonable to expect that the higher the level of
resistance in vitro, the less activity the drug will
have in vivo. However, low level resistance can
also be associated with a poor response.
4.4. Enzymatic-based assays for phenotypic RT
susceptibility testing
RT and protease assays have been widely used
as research tools to measure drug susceptibility
(Ueno et al., 1995; Furline, 1999). However, the
use of these assays for drug susceptibility testing
in plasma has not been possible because of the
low sensitivity of these assays. Recently, several
ultrasensitive RT assays such as PERT and Amp-
RT have been described (Silver et al., 1993; Pyra
et al., 1994; Heneine et al., 1995; Boni et al.,
1996). All these assays use PCR amplification for
the detection of the RT-generated cDNA but
differ in the RNA template and some testing
conditions. We have developed Amp-RT-based
assays for the analysis of RT susceptibility to
RTIs and NNRTIs. The Amp-RT assay detects
 J.G. García-Lerma, W. Heneine / Journal of Clinical Virology 21 (2001) 197–212 205

RT activity by using a known heterologous RNA
template derived from the encephalomyocarditis
virus (EMCV) RNA genome. The RT-derived
EMCV cDNA is detected by PCR amplification
and an ELISA-based hybridization with an inter-
nal EMCV-specific probe (Heneine et al., 1995;
Yamamoto et al., 1996; Garcı́a-Lerma et al.,
1998). Phenotypic resistance to RT inhibitors is
measured by quantitating the RT signal generated
in Amp-RT reactions done in the presence and
absence of the relevant RT inhibitor. Fig. 3 illus-
trates the principle of this testing approach. For
resistant RTs, an Amp-RT signal is seen in RT
reactions done in the presence of drug, while no
detectable RT signal is seen in RTs from sensitive
isolates.
Susceptibility testing of HIV-1 RT by Amp-RT
is done by measuring IC50 and IC90 of the RT
enzyme to NNRTIS or the triphosphate form of
NRTIs. Amp-RT IC50 values determined in sev-
eral wild type and nevirapine- or 3TC-resistant
isolates have been shown to correlate with those
measured by culture-based phenotypic assays
(Garcı́a-Lerma et al., 1999; Vázquez-Rosales et
al., 1999). In contrast to conventional phenotypic
assays, drug susceptibility testing by Amp-RT

Fig. 3. Principle of the Amp-RT-based phenotypic assay for detection of resistance to RT inhibitors. Amp-RT measures the ability
of HIV-1 RT to produce a cDNA copy from an encephalomyocarditis (EMCV) RNA template. HIV-1 RT from plasma is tested
in the absence and presence of several concentrations of drug. The RT-generated EMCV cDNA is detected by PCR amplification
and quantitated by ELISA. IC50 and IC90 for drug are determined and compared with a wild type virus reference virus.
206 J.G. García-Lerma, W. Heneine / Journal of Clinical Virology 21 (2001) 197–212
Fig. 4. Detection of phenotypic resistance to nevirapine in
plasma by Amp-RT (adapted from Vázquez-Rosales et al.,
1999). (A) Level of RT inhibition by 50 mM nevirapine and
correlation with detection of the Y181C mutation by differen-
tial hybridization. Open circles correspond to the RT inhibi-
tion values determined by Amp-RT, and closed circles
correspond to the log10 ratio between mutant genotypes and a
highly conserved wild type region of the HIV-1 RT (MUT/
GNR), (B) Amp-RT IC50 values for nevirapine in sequential
samples from a patient treated with nevirapine. Sequential
samples from days 6, 7, 21, and 28 were tested in the absence
and presence of several concentrations of nevirapine, and
inhibition of RT activity was computed.
does not require virus isolation and culture and,
therefore, provides rapid information (1– 2 days)
on resistance.
A simple testing strategy using one drug con-
centration in the Amp-RT assay was also found
to be useful for rapid screening of resistance in
plasma samples. We have demonstrated the valid-
ity of this testing approach for detecting resistance
to 3TC and nevirapine (Garcı́a-Lerma et al., 1999;
Vázquez-Rosales et al., 1999). Fig. 4 compares
Amp-RT-based phenotypic results with detection
of the Y181C mutation in patients treated with
nevirapine. A correlation between RT susceptibil-
ity by Amp-RT and the levels of Y181C mutation
was found in longitudinally samples collected
from patients receiving nevirapine monotherapy
(Vázquez-Rosales et al., 1999). The decrease in
the level of RT inhibition observed overtime was
also associated with an increase in the Amp-RT
IC50 for nevirapine (Fig. 4). A similar screening
assay was also successfully used for detecting
phenotypic resistance to 3TC in plasma mediated
by the M184V or the MDNR mutations (Garcı́a-
Lerma et al., 1999) (Fig. 5). Both assays have the
ability to detect low proportions ( 10%) of resis-
tant viruses, and therefore, may be used for early
detection of resistance to these drugs (Garcı́a-
Lerma et al., 1999; Vázquez-Rosales et al., 1999)
(Fig. 1a).
The mechanisms of ZDV resistance are not
completely understood and affect the use of the
Amp-RT assay to measure ZDV resistance. RTs
carrying the classical ZDV resistance mutations
(i.e. M41L, T215Y/F, K70R or D67N) have Ki
values or IC50 values for ZDV-TP that are similar
to wild type RTs (Arts and Wainberg, 1996). This
unique enzymatic characteristic precludes the use
of RT assays to detect ZDV resistance. However,
a recent report has shown that efficient discrimi-
nation between wild type and ZDV-resistant RTs
can be achieved enzymatically by adding ATP or
pyrophosphate to the RT reaction (Lennerstrand
et al., 2000). Therefore, using these modified assay
conditions it may be possible to test enzymatically
for ZDV resistance.
Amp-RT-based phenotypic assays provide a
generic tool that can also be used to monitor for
drug resistance in persons infected with highly
divergent viruses. We have found that the re-
bound in RT-based plasma virus load observed in
two HIV-1 group O-infected patients treated with
d4T, 3TC and indinavir, was associated with evi-
dence of phenotypic resistance to Lamivudine by
Amp-RT and with the detection of the M184V
mutation. These Amp-RT-based phenotypic as-
says should therefore facilitate studies aimed at
evaluating the responses of HIV-1 group O-in-
fected patients to RT and protease inhibitors
(Garcı́a-Lerma et al., 2000b).
5. Use of resistance testing for clinical
management
Data from various retrospective and prospec-
tive studies are accumulating to support the use of
drug resistance testing in selecting drugs in many
 J.G. García-Lerma, W. Heneine / Journal of Clinical Virology 21 (2001) 197–212 207

Fig. 5. Detection of phenotypic resistance to 3TC by analysis of RT inhibition by 5 uM 3TC-TP in the Amp-RT assay. Amp-RT
reactions done in the presence and absence of 3TC-TP are shown. Lanes 1 and 2, wild type (wt) HIV-1 RT; Lane 3, 3TC-resistant
(M184V); Lane 4, nevirapine-resistant (181C/Y); lane 5, 3TC/nevirapine-resistant (M184V/YI81C); Lane 6, AZT-resistant (D67N/
K70R/T215F/K219Q); Lanes 7, 8, and 9, HIV-1 RT carrying different multidrug-resistant mutations; Neg, uninfected culture
supernatant.

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